Véronique Dubois

Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Community and Private Health Care Centers

ABSTRACT In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum β-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.

Nosocomial Outbreak Due to a Multiresistant Strain of Pseudomonas aeruginosa P12: Efficacy of Cefepime-Amikacin Therapy and Analysis of β-Lactam Resistance

ABSTRACT Over a 3-year period, 67 patients of the Hospital of Pau (Pau, France), including 64 patients hospitalized in the adult intensive care unit (ICU), were colonized and/or infected by strains ofPseudomonas aeruginosa P12, resistant to all potentially active antibiotics except colistin. Most patients were mechanically ventilated and presented respiratory tract infections. Since cefepime and amikacin were the least inactive antibiotics by MIC determination, all ICU patients were treated with this combination, and most of them benefited. Cefepime-amikacin was found highly synergistic in vitro. Ribotyping and arbitrary primer-PCR analysis confirmed the presence of a single clonal isolate. Isoelectrofocusing revealed that the epidemic strain produced large amounts of the chromosomal cephalosporinase and an additional enzyme with a pI of 5.7, corresponding to PSE-1, as demonstrated by PCR and sequencing. Outer membrane protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the absence of a ca. 46-kDa protein, likely to be OprD, and increased production of two ca. 49- and 50-kDa proteins, consistent with the outer membrane components of the efflux systems, MexAB-OprM and MexEF-OprN. Thus, we report here a nosocomial outbreak due to multiresistant P. aeruginosa P12 exhibiting at least four mechanisms of β-lactam resistance, i.e., production of the penicillinase PSE-1, overproduction of the chromosomal cephalosporinase, loss of OprD, and overexpression of efflux systems, associated with a better activity of cefepime than ceftazidime.

Type II Topoisomerase Quinolone Resistance-Determining Regions of Aeromonas caviae, A. hydrophila, and A. sobria Complexes and Mutations Associated with Quinolone Resistance

ABSTRACT Most Aeromonas strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of Aeromonas caviae (n = 10), A. hydrophila (n = 5), and A. sobria (n = 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the gyrA, gyrB, parC, and parE genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of Aeromonas spp. were highly conserved (≥96.1% identity), despite some genetic polymorphism; they were most closely related to those of Vibrio spp., Pseudomonas spp., and members of the family Enterobacteriaceae (72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-83→Ile (10 strains) or Ser-83→Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80→Ile (three strains) or Ser-80→Arg change, or at position 84, yielding a Glu-84→Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double gyrA-parC missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single gyrA mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic Aeromonas spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.

High Genetic Stability of Integrons in Clinical Isolates of Shigella spp. of Worldwide Origin

ABSTRACT Over a 12-year period, 68 Shigella strains (31 S. sonnei, 30 S. flexneri, 4 S. dysenteriae, and 3 S. boydii strains) were collected in a French University Hospital from the stools of patients who generally had a recent history of travel to various parts of the world (91%), particularly Africa (67%). These strains were often resistant (streptomycin, spectinomycin, trimethoprim, tetracycline, and sulfonamides, 66 to 84%; ampicillin and chloramphenicol, 34 to 38%; nalidixic acid, 4%) and even multiresistant (87%), and they generally carried integrons (81%) of class 1 (21%), class 2 (47%), or both (13%). Class 1 integrons were associated with ampicillin resistance due to the production of an OXA-30 β-lactamase in S. flexneri and S. dysenteriae. Class 2 integrons were associated with trimethoprim resistance in S. sonnei. Class 1 and class 2 integrons were inserted within transposons Tn21 and Tn7, respectively, themselves located on the bacterial chromosome, except in one strain. Class 1 integrons showed an atypical organization consisting of the insertion sequence IS1 at the 3′ end instead of the typical 3′ conserved segment and two blaOXA-30 and aadA1 gene cassettes, despite the absence of epidemiological relationships between the strains, and an apparently functional integrase. Class 2 integrons showed the same albeit classical organization with the three dfrA1, sat, and aadA1 gene cassettes. Occasionally, the 3′ end was deleted and the aadA1 gene cassette was unexpressed. Thus, integrons contributed only in part to the multidrug resistance of the Shigella strains. The highly conserved organization of integrons might be related to their location within mobile genetic superstructures.

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments.

ABSTRACT A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

Occurrence of Listeria spp. in Effluents of French Urban Wastewater Treatment Plants

ABSTRACT Listeria spp. were found in most treated waters (84.4%) and raw sludge (89.2%) of six French urban wastewater treatment plants and one composting facility, examined monthly over a 1-year period. Most strains belonged to Listeria monocytogenes, serotypes 4b/4e being predominant. Sludge composting and liming reduced or prevented Listeria contamination.

Clinical and Molecular Analysis of Extended-Spectrum β-Lactamase-Producing Enterobacteria in the Community Setting

ABSTRACT During a previous survey, five extended-spectrum β-lactamase (ESBL)-producing enterobacteria (ESBLE) (two Enterobacter aerogenes isolates expressing TEM-24b, two Escherichia coli isolates expressing TEM-21 or TEM-24b, and one Klebsiella pneumoniae isolate expressing SHV-4/TEM-15) responsible for urinary tract infections (UTIs) were found among 1,584 strains collected from community patients. The aim of the present study was to elucidate the route of emergence of these typically nosocomial organisms in the community. Thus, the files of the five patients were analyzed over at least a decade, and potentially related ESBLE from hospitals or clinics were examined. Their enzymes were characterized at a molecular level, and the strains were typed by amplified-primed PCR, enterobacterial repetitive intergenic consensus PCR, and restriction plasmid profile. All patients (C1 to C5) had risk factors for ESBLE acquisition, including past history of hospitalization (2.5 to 23 months before). Four (C1 and C3 to C5) had previously received antibiotics (concomitantly to 35 months earlier), two (C1 and C3) had indwelling urinary catheters and recurrent UTIs, and three (C2, C3, and C5) formerly experienced ESBLE-induced UTIs (2 to 11 months before). The same ESBLE and/or an identical or similar ESBL-encoding plasmid was identified in the hospital ward (C1 to C4) or in a clinic (C5) where the patients had previously resided. Patients C1 and C4, infected with different ESBLE carrying a closely related plasmid, were hospitalized in the same unit. Persistence of ESBLE over at least a 5-year period was demonstrated for patient C3. Thus, community-acquired UTIs in these patients likely resulted from nosocomially acquired ESBLE or an ESBL-encoding plasmid followed by a prolonged digestive carriage.

Prolonged Outbreak of Infection Due to TEM-21-Producing Strains of Pseudomonas aeruginosa and Enterobacteria in a Nursing Home

ABSTRACT Over a 6-year period, 24 extended-spectrum β-lactamase (ESBL)-producing isolates of Pseudomonas aeruginosa were collected from 18 patients living in a nursing home. These isolates had a delayed development of a red pigment and exhibited a similar antibiotype (resistance to all β-lactams except for imipenem and to gentamicin, tobramycin, netilmicin, ciprofloxacin, and rifampin) associated with the production of the TEM-21 β-lactamase and a type II 3′-N-aminoglycoside acetyltransferase [AAC(3)-II] enzyme. Surprisingly, serotyping showed that these isolates belonged to four successive serotypes (P2, P16, P1, and PME), although molecular typing by PCR methods and pulsed-field gel electrophoresis yielded identical or similar profiles. Moreover, in all isolates the blaTEM-21 gene was part of a chromosomally located Tn801 transposon truncated by an IS6100 element inserted within the resolvase gene, and the aac(3)-II gene was adjacent to this structure. During the same period, 17 ESBL-producing isolates of enterobacteria were also collected from 10 of these patients. These isolates harbored a similar large plasmid that contained the blaTEM-21 and the aac(3)-II genes and that conferred additional resistance to sulfonamides and chloramphenicol, as well as to kanamycin, tobramycin, netilmicin, and amikacin, conveyed by an AAC(6′)-I enzyme. The blaTEM-21 gene was part of the Tn801 transposon disrupted by IS4321. Thus, a single clone of P. aeruginosa that had undergone a progressive genetic drift associated with a change in serotype appeared to be responsible for an outbreak of nosocomial infections in a nursing home. This strain has probably acquired the blaTEM-21-encoding plasmid that was epidemic among the enterobacteria at the institution, followed by chromosomal integration and genomic reorganization.

Clinical Strain of Pseudomonas aeruginosa Carrying a blaTEM-21 Gene Located on a Chromosomal Interrupted TnA Type Transposon

ABSTRACT A clinical isolate of Pseudomonas aeruginosa was found to produce a clavulanic acid-inhibited extended-spectrum β-lactamase with a pI of 6.4. PCR, cloning, and sequencing experiments showed that the corresponding blaTEM-21 gene was part of a chromosomally located Tn801 transposon disrupted by an IS6100 element and adjacent to an aac(3)-II gene.

CTX-M-producing Escherichia coli in a maternity ward: a likely community importation and evidence of mother-to-neonate transmission

OBJECTIVES To investigate the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing strains of Escherichia coli (4%, 10/250 consecutive isolates) recovered during a 5 month period in the maternity ward of the University Hospital of Bordeaux, France. METHODS beta-Lactam resistance transfer was analysed by conjugation and transformation. ESBLs were characterized by isoelectric focusing, PCR amplification and sequencing. The relatedness of the strains was examined by PFGE and phylogenetic group determination. Plasmids were characterized by incompatibility group and restriction analysis. RESULTS Ten ESBL-producing E. coli were isolated from urinary or genital samples of eight mothers and from gastric fluids of two newborns of carrier mothers. The patients were hospitalized in five different units of the maternity ward. Transconjugants, obtained for 7 of the 10 strains, and wild-type strains exhibited various antibiotypes. Different CTX-M enzymes were characterized: CTX-M-1 (n = 4); CTX-M-14 (n = 3); CTX-M-32 (n = 2); and CTX-M-28 (n = 1). The strains recovered from two mothers and their respective babies were identical. All the other strains were epidemiologically unrelated. Furthermore, various plasmids were identified. Environmental samples from the common echographic and sampling rooms did not reveal the presence of ESBL-producing enterobacteria. CONCLUSIONS The data argue against the occurrence of a nosocomial outbreak and support the hypothesis of an importation of community-acquired ESBL-producing strains into the hospital through colonized/infected patients. At present, not only patients transferred from other hospitals or long-term care facilities are at risk of carrying ESBL-producing enterobacteria on hospital admission, but also community patients.